title: Cbl ubiquitination of p85 is essential for Epo-induced EpoR endocytosis.
abstract: Erythropoietin (Epo) binding to the Epo receptor (EpoR) elicits downstream signaling that is essential for red blood cell production. One important negative regulatory mechanism to terminate Epo signaling is Epo-induced EpoR endocytosis and degradation. Defects in this mechanism play a key role in the overproduction of erythrocytes in primary familial and congenital polycythemia (PFCP). Here we have identified a novel mechanism mediating Epo-dependent EpoR internalization. Epo induces Cbl-dependent ubiquitination of the p85 regulatory subunit of PI3K, which binds to phosphotyrosines on EpoR. Ubiquitination allows p85 to interact with the endocytic protein epsin-1, thereby driving EpoR endocytosis. Knockdown of Cbl, expression of its dominant negative forms, or expression of an epsin-1 mutant devoid of ubiquitin-interacting motifs all compromise Epo-induced EpoR internalization. Mutated EpoRs mimicking those from PFCP patients cannot bind p85, co-localize with epsin-1, or internalize on Epo stimulation and exhibit Epo hypersensitivity. Similarly, knockdown of Cbl also causes Epo hypersensitivity in primary erythroid progenitors. Restoring p85 binding to PFCP receptors rescues Epo-induced epsin-1 co-localization and EpoR internalization and normalizes Epo hypersensitivity. Our results uncover a novel Cbl/p85/epsin-1 pathway in EpoR endocytosis and show that defects in this pathway contribute to excessive Epo signaling and erythroid hyperproliferation in PFCP.
authors.affiliation: Department of Cell Biology, University of Texas Southwestern Medical Center, Dallas, TX.
authors.forename: Gamze B
authors.initials: GB
authors.lastname: Bulut
authors.forename: Rita
authors.initials: R
authors.lastname: Sulahian
authors.forename: Huiyu
authors.initials: H
authors.lastname: Yao
authors.forename: Lily Jun-shen
authors.initials: LJ
authors.lastname: Huang
mesh_terms: D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002478:Cells, Cultured; D058543:Class Ia Phosphatidylinositol 3-Kinase / Q000235:genetics / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects*; D004921:Erythropoietin / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D051379:Mice; D018345:Mice, Knockout; D009154:Mutation; D011086:Polycythemia / Q000151:congenital / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding / Q000187:drug effects; D050721:Proto-Oncogene Proteins c-cbl / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000235:genetics; D054875:Ubiquitination / Q000187:drug effects
chemical_list: D033942:Adaptor Proteins, Vesicular Transport; D017467:Receptors, Erythropoietin; C114154:epsin; D004921:Erythropoietin; D050721:Proto-Oncogene Proteins c-cbl; D058543:Class Ia Phosphatidylinositol 3-Kinase; C493849:Cbl protein, mouse